Growth inhibition of bovine pulmonary artery smooth muscle cells following long-term heparin treatment.

Heparin (HP) inhibits pulmonary artery smooth muscle cell (PASMC) growth in vitro and vascular remodeling in vivo. Bârzu et al. (1994) suggested that the antiproliferative effect of HP on rat aortic smooth muscle cell in vitro diminishes with prolonged exposure to heparin. We exposed cultured bovine PASMC (BPASMC) to prolonged pretreatment with 20 microg/ml of 0-hexanoylated HP from passages 3 to13 and compared them to control (no pretreatment) cultures of identical passages. The pretreated BPASMC and control groups were growth arrested for 48 h, followed by treatment of 0-hexanoylated HP at different doses. On day 5, the growth inhibition of BPASMC was determined. The percent inhibition by 1 microg/ml of 0-hexanoylated HP was 46 +/- 14% versus 62 +/- 13%, for control and pretreated BPASMC, respectively. At 10 microg/ml the inhibition was 62 +/- 7% versus 84 +/- 6%. For 100 microg/ml the inhibition increased to 92 +/- 5% versus 100% and at 200 microg/ml the inhibition was 95 +/- 3% versus 100%. BPASMC (with or without preexposure to 0-hexanoylated HP), at passage 13, were sensitive to the growth inhibitory effect of 0-hexanoylated HP with no significant difference among the groups (95 +/- 3% inhibition vs. 100% for pretreated BPASMC). We found that 0-hexanoylated HP-induced necrosis as shown by flow cytometry and only minor apoptosis. Caspase-3 and PARP detection was insignificant between the groups. In summary, no cell subpopulation at long-term treatment exhibited resistance to 0-hexanoylated HP. The HP antiproliferative effect on SMC is potentially important in defining new approaches to the treatment of the remodeled vasculature of pulmonary hypertension. Liss, Inc.

Bladder cancer therapy using combined proliferating cell nuclear antigen antisense oligonucleotides and recombinant adenovirus p53.

OBJECTIVE: To evaluate the antitumor efficacy of proliferating cell nuclear antigen antisense oligonucleotide (PCNA-ASO) in combination with recombinant adenovirus p53 (Ad-p53) against bladder cancer EJ and BIU-87 cells in vitro and in vivo. METHODS: Cells were transfected with Ad-p53 (100 MOI), and PCNA-ASO (1.6 micro mol/L) was then introduced into the cells using a cationic lipid (lipofectamine, 20 micro l/ml). In vitro and in vivo antitumor effects of combining PCNA-ASO with Ad-p53 were measured using the MTT assay, flow cytometry, clone formation, and a nude mice model. RESULTS: The combination of PCNA-ASO and Ad-p53 inhibited cell viability in both the EJ (89.3%) and BIU-87 (78.6%) cell lines. The ability of the cells to form foci was also reduced by 74.8% in EJ cells and by 67.5% in BIU-87 cells (P < 0.01). A significant decrease of cells in the S phase (11.4% in EJ cells, 14.6% in BIU-87 cells) and a significant increase of cells in G1 phase (62.2% in EJ, 56.8% in BIU-87) were noted. The mean tumor volume after 7 days of treatment with PCNA-ASO or Ad-p53 in combination decreased to 47.6% or 36.4% of the initial tumor size in the two cell lines respectively. CONCLUSION: These results indicate that combined PCNA-ASO and Ad-p53 in the treatment of bladder cancer with mutant p53 has important therapeutic potential, significantly suppressing the growth of human bladder cancer both in vitro and in vivo.